This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The COBRE Imaging and Physiology Core provides a wide range of imaging techniques for fixed and live-cell immunofluorescence preparations. We maintain a Noran/Prairie Technologies LSCM (laser scanning confocal microscope), a DeltaVision RT restoration microscopy system, a Zeiss/BioRad dedicated multiphoton LSCM, a Nikon TIRF (total-internal reflection fluorescence) system, a Lamda DG-4 ratiometric imaging system, an Andor iXon/Olympus widefield fluorescence microscope, and a Nikon SMZ-1500 fluorescence stereoscope. Patch-clamp electronics are available for use on most microscope systems, and each system has an integrated perfusion setup. The Noran is capable of high-speed image acquisition and is used primarily for calcium imaging on whole tissues and isolated cells. The DeltaVision system has a temperature and CO2-controlled incubator allowing for time-lapse experiments on cultured cells, and also excels at immunofluorescence and colocalization experiments on fixed samples. The BioRad Radiance 2100 Dedicated Multiphoton features a Coherent Chameleon femtosecond pulsed IR laser and a fixed-stage upright Olympus microscope. The pulsed infrared excitation, coupled with non-descanned detectors, make it possible to image at depths of up to 500 microns for several hours. It is currently being used for calcium-imaging and uncaging experiments in brain-slices as well as retrograde labeling of neurons in the spinal cord of developing chick embryos. The ratiometric imaging system is used for imaging of fura-2 loaded cells or cells transfected with the FRET-based calcium indicator Cameleon. The TIRF system is used in vesicular trafficking studies as well as cell adhesion and migration experiments. Currently, it is being set up for calcium imaging near the cell membrane on patch-clamped rodent cells. The Andor iXon system is a high-speed EM CCD coupled with an Olympus microscope, designed to image fast calcium events in tissue preparations. For data deconvolution and analysis, we have two dedicated workstations running softWoRx (Applied Precision) for deconvolution, MetaMorph (Universal Imaging), Volocity (Perkin Elmer) and ImageJ (NIH) for general analysis and rendering. The EM CCD workstation includes Andor iQ-CORE analysis software. Equipment scheduling is online, and user hours are recorded in the core database.